Functional Impairment of Myeloid Dendritic Cells during Advanced Stage of HIV-1 Infection: Role of Factors Regulating Cytokine Signaling

Introduction

Severely immunocompromised state during advanced stage of HIV-1 infection has been linked to functionally defective antigen presentation by dendritic cells (DCs). The molecular mechanisms behind DC impairment are still obscure. We investigated changes in DC function and association of key regulators of cytokine signaling during different stages of HIV-1 infection and following antiretroviral therapy (ART).

Methods

Phenotypic and functional characteristics of circulating myeloid DCs (mDCs) in 56 ART-naive patients (23 in early and 33 in advanced stage of disease), 36 on ART and 24 healthy controls were evaluated. Sixteen patients were studied longitudinally prior-to and 6 months after the start of ART. For functional studies, monocyte-derived DCs (Mo-DCs) were evaluated for endocytosis, allo-stimulation and cytokine secretion. The expression of suppressor of cytokine signaling (SOCS)-1 and other regulators of cytokine signaling was evaluated by real-time RT-PCR.

Results

The ability to respond to an antigenic stimulation was severely impaired in patients in advanced HIV-1 disease which showed partial recovery in the treated group. Mo-DCs from patients with advanced HIV-disease remained immature with low allo-stimulation and reduced cytokine secretion even after TLR-4 mediated stimulation ex-vivo. The cells had an increased expression of negative regulatory factors like SOCS-1, SOCS-3, SH2-containing phosphatase(SHP)-1 and a reduced expression of positive regulators like Janus kinase(JAK)2 and Nuclear factor kappa-light-chain-enhancer of activated B cells(NF-κB)1. A functional recovery after siRNA mediated silencing of SOCS-1 in these mo-DCs confirms the role of negative regulatory factors in functional impairment of these cells.

Conclusions

Functionally defective DCs in advanced stage of HIV-1 infection seems to be due to imbalanced state of negative and positive regulatory gene expression. Whether this is a cause or effect of increased viral replication at this stage of disease, needs further investigation. The information may be useful in design of novel therapeutic targets for better management of disease.     

Identification of cultivation condition to produce correctly folded form of a malaria vaccine based on Plasmodium falciparum merozoite surface protein-1 in Escherichia coli

The C-terminal, 19-kDa domain of Plasmodium falciparum merozoite surface protein-1 (PfMSP-1₁₉) is among the leading vaccine candidate for malaria due to its essential role in erythrocyte invasion by the parasite. We designed a synthetic gene for optimal expression of recombinant PfMSP-1₁₉ in Escherichia coli and developed a scalable process to obtain high-quality PfMSP-1₁₉. The synthetic gene construct yielded a fourfold higher expression level of PfMSP-1₁₉ in comparison to the native gene construct. Optimization of cultivation conditions in the bioreactor indicated important role of yeast extract and substrate feeding strategy for obtaining enhanced expression of soluble and correctly folded PfMSP-1₁₉. It was observed that the higher expression level of PfMSP-1₁₉ was essentially associated with the generation of higher level of incorrectly folded PfMSP-1₁₉. A simple purification procedure comprising metal affinity and ion exchange chromatography was developed to purify correctly folded form of PfMSP-1₁₉ from cell lysate. Biochemical and biophysical characterization of purified PfMSP-1₁₉ suggested that it was highly pure, homogeneous, and correctly folded.     

Detection and confirmation of Clostridium botulinum in water used for cooling at a plant producing low-acid canned foods

Our laboratory tested water samples used for cooling low-acid canned foods at a canning facility under investigation by the U.S. Food and Drug Administration. We used an enzyme-linked immunosorbent assay with digoxigenin-labeled antibodies (DIG-ELISA) and real-time PCR as screening methods and confirmed the presence of neurotoxin-producing Clostridium botulinum in the samples by mouse bioassay.     

Antiplasmodial and cytotoxicity evaluation of 3-functionalized 2-azetidinone derivatives

3-Azido-, 3-amino- and 3-(1,2,3-triazol-1-yl)-β-lactams were synthesized and evaluated for their antiplasmodial activity against four strains of Plasmodium falciparum and KB cells for their cytotoxicity profiles. The presence of a cyclohexyl substituent at N-1 and a phenyl group on the triazole ring markedly improved the activity profiles of triazole-tethered β-lactam exhibiting IC₅₀ values of 1.13, 1.21 and 1.00 μM against 3D7, K1 and W2 strains respectively.     

Crystal structure of a substrate complex of Mycobacterium tuberculosis beta-ketoacyl-acyl carrier protein synthase III (FabH) with lauroyl-coenzyme A

Beta-ketoacyl-acyl carrier protein synthase III (FabH) catalyzes a two step reaction that initiates the pathway of fatty acid biosynthesis in plants and bacteria. In Mycobacterium tuberculosis, FabH catalyzes extension of lauroyl, myristoyl and palmitoyl groups from which cell wall mycolic acids of the bacterium are formed. The first step of the reaction is an acyl group transfer from acyl-coenzyme A to the active-site cysteine of the enzyme; the second step is acyl chain extension by two carbon atoms through Claisen condensation with malonyl-acyl carrier protein. We have previously determined the crystal structure of a type II, dissociated M.tuberculosis FabH, which catalyzes extension of lauroyl, myristoyl and palmitoyl groups. Here we describe the first long-chain Michaelis substrate complex of a FabH, that of lauroyl-coenzyme A with a catalytically disabled Cys-->Ala mutant of M.tuberculosis FabH. An elongated channel extending from the mutated active-site cysteine defines the acyl group binding locus that confers unique acyl substrate specificity on M.tuberculosis FabH. CoA lies in a second channel, bound primarily through interactions of its nucleotide group at the enzyme surface. The apparent weak association of CoA in this complex may play a role in the binding and dissociation of long chain acyl-CoA substrates and products and poses questions pertinent to the mechanism of this enzyme.     

Enhanced expressions of endometrial tumour necrosis factor alpha and its receptors during early pregnancy in bonnet monkeys

Tumour necrosis factor alpha (TNF-alpha), a pro-inflammatory cytokine may play an active role in stimulating inflammatory reactions during pregnancy. However, the expression of endometrial TNF-alpha has not been investigated especially during early pregnancy, a phenomenon invariably accompanied by inflammatory reaction. In the present study, the endometrial expressions of TNF-alpha and its receptors (TNFR1 and TNFR2) during early pregnancy, when the embryo lies free in the zona hatched state in the uterine lumen, were analyzed by immunohistochemistry. The endometrial expressions of TNF-alpha, TNFR1 and TNFR2 were found to be significantly up-regulated (p < 0.05) in the glandular epithelium on day 6 post-ovulation in pregnant animals. The alteration in the expression of these molecules may contribute to the induction of local inflammatory reactions during implantation.     

Molecular heterogeneity in Yersinia enterocolitica and 'Y. enterocolitica-like' species--Implications for epidemiology, typing and taxonomy

Yersinia enterocolitica is an extremely heterogeneous species. Serotyping and biotyping have been used extensively, in the past, to study its heterogeneity and epidemiology. Application of methods like ribotyping, pulsed-field gel electrophoresis and a host of other genomic techniques have further revealed molecular heterogeneity in this species. Furthermore, these methods may be used effectively to supplement serotyping and biotyping schema for studying epidemiology of Y. enterocolitica. This is evident from the ability of some of these methods to subtype strains belonging to serogroups O:3, O:9 and O:8 - which are most commonly encountered in human Yersiniosis. Multilocus enzyme electrophoresis and nucleotide sequencing have reiterated the taxonomic relationships of this organism. However there is paucity of information about the molecular heterogeneity of 'Y. enterocolitica-like' species, which need to be addressed in the future. Also, newer techniques such as amplified fragment length polymorphism, VNTR-based typing and multilocus sequence typing should be applied to further understand epidemiology, population structure and evolutionary genetics of Y. enterocolitica and 'Y. enterocolitica-like' species.     

Haptoglobin polymorphism among fourteen populations of India

Haptoglobin (HP) is a serum protein that has the capability of binding the extracorpuscular haemoglobin released during haemolysis. It plays an important role in protection of haemolytic disease by reducing the oxidative and peroxidative potential at free haemoglobin. The present study was aimed to determine the prevalence of HP polymorphism among different Indian populations, anthropologically belonging to diverse ethnicity. The polymorphism was screened among 642 unrelated individuals belonging to 14 population groups of India including both tribal and non-tribal caste groups from different geographical regions of India with distinct linguistic affiliations. An attempt is also made to understand the distribution of HP polymorphism among the studied populations. The result reveals the HP gene to be polymorphic in all the studied populations. Except the two tribal populations (Thotis of Andhra Pradesh and Patelias of Rajasthan) and one caste population (Rajput of Himachal Pradesh), all the studied populations are found to obey the Hardy-Weinberg equilibrium. The significance of the present study is elucidated with the prevalence of high mutant HP*2 allele frequency in India. Selection could be one of the most plausible explanations for this high HP frequency because of its uniformly high occurrence among all the studied populations.     

Native X-DING-CD4 protein secreted by HIV-1 resistant CD4+ T cells blocks activity of IL-8 promoter in human endothelial cells infected with enteric bacteria

Onsets of bacterial infections devastate the compromised immune system in AIDS patients. Damaged gut mucosa permits dissemination of bacterial toxins into deeper layers and hyper-activation of the immune system. We previously reported that the unfractionated supernatants of HIV-resistant CD4(+) T cells impeded the NF-κB/DNA binding in macrophages induced by either HIV-1 or LPS. The active component of this soluble material was identified as X-DING-CD4 (extracellular DING from CD4 T cells). We hypothesized that the anti-inflammatory effect of the X-DING-CD4 protein might extend to non-immune cells, for example endothelial cells, undergoing persistent endotoxin stimulation in the course of advanced HIV disease. To test this proposition, we evaluated the efficiency of NF-κB and Ap-1 binding to the IL-8 promoter in LPS-activated endothelial cells and control human macrophages exposed to native X-DING-CD4 protein. We found a deficiency of NF-κB- but not AP-1-DNA binding in the systems where cells were treated with native soluble X-DING-CD4 protein. The X-DING-CD4-mediated inhibition of the IL-8 promoter also resulted in a reduction of the soluble IL-8 protein in endothelial cells and human macrophages infected with a subset of enteric bacteria frequently causing diarrhea in progressive HIV disease. Bacterial endotoxin did not induce the endogenous X-DING-CD4 mRNA activity in human macrophages and transformed CD4(+)T cells, indicating that the reduction of LPS-mediated IL-8 promoter activation was not related to de novo X-DING-CD4 protein synthesis, but depended on function of the exogenous X-DING-CD4 protein. This study provides evidence that the X-DING-CD4 protein might be developed as a novel biotherapeutic to control LPS-mediated inflammation in advanced HIV disease.